The goal of the proposed research is to develop the use of single-stranded oligodeoxynucleotides (ssODN) to repair single-base mutations in melanocytes and keratinocytes in tissue culture and in animal. This competitive renewal is based on our three new findings: (1) ssODN has a similar capability of gene correction as the RNA-DNA oligonucleotide (RDO). ssODN is much easier to synthesize and purify than RDO for practical application. (2) Our preliminary results indicate simultaneous targeting of two genes is feasible in melanocytes and occurs with a relatively high frequency, 25%. We hypothesize that use of a combination of two ssODNs, one targeting the gene of interest and the other one targeting a defective selective marker, may provide a general selection method and overcome the low frequency, a main limitation in the ODN-based gene targeting. A unique advantage of albino melanocytes is that tyrosinase gene correction can be detected as a viable pigmentation change. In Aims 1 and 2, we will further develop the ssODN gene repair strategy in melanocytes in tissue culture and in animal. To link the gene correction to epidermis, we will carry out in vivo application of ssODN by using the albino mouse expressing the stem cell factor under the control of K14 promoter. These mice express a large number of melanocytes in epidermis and dermis and will provide a pigmentation change upon gene correction that can be easily monitored. Thus, Aim 1 and 2 will use the same system that we have already successfully developed and link it to the epidermal gene correction. (3) Our preliminary results suggest that keratinocytes in murine skin may have a higher rate of gene correction than those in tissue culture. We hypothesize that ODN may be capable of gene correction of epidermal stem cells and a high turnover rate of epidermis may actually amplify the low frequency. We also hypothesize that a low level of gene repair may be sufficient for healing of blisters because the corrected keratinocytes will have selective advantage over the mutated cells. We will test the feasibility of the ODN-based gene repair to heal or reduce the blister in the mouse containing a dominant mutation in COL7A1, which will be generated in the Project 4. Our in vivo results and accessibility of the tissue make compelling bases for in situ application of ssODN to skin. If successful, this approach will have a potential clinical application of ODNs to severely affected areas of EB patient.